The invention relates to techniques for the separation, purification, or both, of biolgical materials and, more specifically, to a method and apparatus for isoelectric focusing and isotachophoresis.
Isoelectric focusing, isotachophoresis, and zone electrophoresis are variants of electrophoretic techniques, differing in the buffer system employed and mode of separation achieved. The theoretical distinction of three methods has been described in some detail in Bier, 219 Science 1281-87 (1983).
Zone Electrophoresis ("ZE"), is the oldest of these techniques and most commonly used. Separation is carried out in the presence of a background of homogeneous buffer, and sample components separate according to their mobilities in this buffer. No steady state is ever reached, but migration continues with gradual broadening of sample zones due to diffusion and other effects.
Isotachophoresis ("ITP") is a more recent variant of electrophoresis, characterized by the fact that separation is carried out in a discontinuous buffer system. Sample material to be separated is inserted between a "leading electrolyte" and a "terminating electrolyte", the characteristic of these two buffers being that the leader has to have ions of net mobility higher than those of sample ions, while the terminator must have ions of net mobilities lower than those of sample ions. In such a system, sample components sort themselves according to decreasing mobilities from leader to terminator, in a complex pattern governed by the so-called Kohlrausch regulating function. The process has been described repeatedly, as for instance, Bier and Allgyer, Electrokinetic Separation Methods 443-69 (Elsevier/North-Holland 1979).
It is further characteristic of ITP that a steady state is eventually reached, where all components migrate at same velocity (hence the name) in sharply defined contiguous zones. Sample components can be separated in such a contiguous train of components by insertion of "spacers" with mobilities intermediary between those of the components one wishes to separate.
Isoelectric focusing ("IEF"), also sometimes called electrofocusing, is a powerful variant of electrophoresis. The principle of IEF is based on the fact that proteins and peptides, as well as most biomaterials, are amphoteric in nature, i.e., are positively charge in acid media and negatively charged in basic media. At a particular pH value, called the isoelectric point (PI), there is reversal of net charge polarity, the biomaterials acquiring zero net charge.
If such amphoteric materials are exposed to a d.c. current of proper polarity in a medium exhibiting a pH gradient, they will migrate, i.e., `focus` toward the pH region of their PI, where they become virtually immobilized. Thus a stationary steady state is generated, where all components of the mixture have focused to their respective PIs.
The pH gradient is mostly generated `naturally` i.e, through the electric current itself. Appropriate buffer systems have been developed for this purpose, containing amphoteric components which themselves focus to their respective PI values, thereby buffering the pH of the medium.
Such buffer mixtures are known as `carrier ampholytes`, the best known being "Ampholine", a trademark of the LKB Produkter AB, a Swedish company. Other carrier ampholyte mixtures can be formulated by judicious mixing of suitable ampholytes, as, for example, described in Bier, 211 J. Chromatography 313-35 (1981).
The two variants, IEF and ITP, differ in that IEF attains a stationary steady state whereas in ITP a migrating steady state is obtained. Thus, in IEF a finite length of migrating channel is always sufficient. In ITP, complete resolution may require longer migrating channels than is practical. In such case, the migrating components can be virtually immobilized by applying a counterflow of leading electrolyte, the rate of counterflow being matched to the rate of frontal migration of the sample ions. This is also known in the art.
IEF is most frequently carried out in polyacrylamide or agarose gels, where all fluid flow disturbances are minimized. ITP is most often carried out in capillaries. The sample is inserted at one end of the capillary, at the interface between leader and terminator, and the migration of separated components recorded by appropriate sensors at the other end of the capillary. Both such systems are used mainly for analytical or micro-preparative purposes.
The scaling up of any electrophoretic technique is difficult because of the need to stabilize the fluid system against convection. The easiest fluid stabilization is achieved in gels or with other supporting media, such as granulated beds, etc. Unfortunately, such stabilized systems do not lend themselves to separations involving flow of process fluid, yet such flow, whether continuous or recycling, appears to be the best approach for increasing the capacity of the techniques. Continuous flow is best carried out in free fluids, unsupported by gels or granulated beds. Separations in free fluids require stabilization against flow disturbances. These disturbances could disrupt the orderly separation of sample components. The need for fluid stabilization is well recognized by practitioners of the art and, to achieve it, a variety of principles have been utilized and incorporated into diverse instruments.
One of the most common principles utilized for flow stabilization is confinement of the process fluid, i.e. carrier buffer and sample solution, to a narrow liquid film contained within a channel between two parallel plates. Within the channel it is generally assumed that viscous forces maintain fluid stability. Numerous such instruments have been designed and patented for continuous flow electrophoresis.
In such continuous flow instruments the d.c. electric field is applied in a direction perpendicular to buffer and sample flow. The critical feature of such instruments seems to be the dimension of the gap between the two parallel plates, i.e., the thickness of the fluid film. This is usually of the order of 0.5 to 1.5 mm. The passage of the electric current generates heat, and thus one or both of the parallel plates are cooled. The cooling capacity of these plates sets the limit for power dissipation within the apparatus. It is implicit in such continuous flow devices, whether applied to ZE, IEF, or ITP, that separation of sample functions be obtained in a single pass through the apparatus. This requires slow flow of buffer and long residence time of the sample within the apparatus.
While such instruments are in reasonably wide use, their operation is limited by several factors. Only very dilute solutions can be utilized, of the order of 0.01 to 0.2% solute concentration, otherwise density gradients between sample and carrier buffer may cause convective disturbances. Three other factors disturb separation: (1) electroosmosis causes a parabolic flow of liquid in the plane of the electric field, electroosmosis being due to the electric charge at the inner surface of the parallel plates; (2) the downward flow of the liquid through the narrow gap also causes a parabolic flow velocity profile in a direction perpendicular to that due to electroosmosis. Thus, the residence time of the fluid in the center of the gap is much shorter than that of the fluid close to the wall; (3) finally, as the fluid at the center of the gap is warmer than at the walls, all electrophoretic parameters (conductivity, viscosity, electrical field, mobility of ions, etc.) are affected. The effects of the three factors are complex and cause the well known `crescent phenomenon` (Strickler and Sacks, 209 Annals New York Acad. Sci. 497-514 (1973)), i.e., a crescent-like deformation of the migrating sample zones. The crescent deformation is most pronounced closest to the walls of the electrophoretic chamber. To minimize it, the sample stream is mostly injected only into the center of the gap, thus seriously limiting the throughput capacity of the apparatus. Of all these factors, gravity was assumed to be the most important limitation on the performance of the instruments. It is for this reason that McDonnell Douglas Astronautics Co. has constructed and tested is well publicized continuous flow apparatus specifically designed for operation in the reduced gravity of orbiting spacecraft.
All present instruments of the continuous flow kind were designed and are applied principally to ZE. The object of the present invention is to demonstrate how these and other difficulties with current instruments can be avoided. The invention is restricted only to IEF and ITP, where steady states are achieved, and is not applicable to ZE. Thus, the apparatus and method which are objects of the present invention may be considered as the first ones specifically designed for IEF and ITP.